DESCRIPTION: To understand the functions of proteins at the molecular level, it is necessary to have practical means for probing their structural and dynamical features. The rates at which peptide amide hydrogens undergo isotopic exchange when proteins are placed in D(2)O has been used extensively as a probe of protein structure and dynamics. Research currently funded through this grant has demonstrated that amide hydrogen exchange rates can be determined with high accuracy by mass spectrometry, and that this approach offers several advantages over other methods currently used to quantify hydrogen exchange in proteins. For example, amide hydrogen exchange rates can be determined for proteins of virtually any size, and typically requires only picomoles of protein for an analysis. Hydrogen exchange occurring on the time scale from milliseconds to months can be quantified by the protein fragmentation/MS method developed in the current program. Following a period of deuterium exchange in, the pH is decreased to 2-3 to quench the hydrogen exchange reaction. Pepsin is added to fragment the protein into short peptides whose molecular weights are determined by directly-coupled HPLC electrospray ionization mass spectrometry. The deuterium contents of the peptides are determined from their molecular weights. The longterm goal of the proposed investigation is to further develop the protein fragmentation/MS method so that it will become a mainstream tool for investigations of protein structure and dynamics. To achieve this goal, the protein fragmentation/MS method will be adapted to specific types of applications for which amide hydrogen exchange has already proved useful. Four different types of applications have been proposed to demonstrate that the protein fragmentation/MS approach can be used advantageously to investigate protein structure and dynamics. These applications include [1] a study of the thermodynamics and kinetics of protein unfolding, [2] a study of membrane-bound proteins designed to detect conformational changes occurring while the protein is bound to the membrane, [3] a study designed to identify structures of protein folding intermediates which will illuminate mechanisms through which proteins fold, and [4] a study designed to characterize structural changes occurring in a very large protein (M(r)124,000). In addition, several projects have been proposed to improve the sensitivity, speed and spatial resolution of the protein fragmentation/MS method. It is important to note that existing methods for measuring amide hydrogen exchange rates in proteins are not suited for any of the proposed applications.